This invention relates to a method of and apparatus for determining the normality of biological cells.
In reference (1), which is hereby incorporated by reference, each cell of a population of biological cells, labelled with a lipid soluble fluorescent dye, is simultaneously excited by a beam of polarized light. Fluorescence emitted by the population is, itself, polarized; and the average state of polarization of this fluorescence is determined by the intensities of fluorescence, polarized parallel and perpendicular to the direction of polarization of the excitation beam. An analytical combination of the intensities determines the average state of fluorescent polarization in terms of fluidity, degree of fluorescent polarization, microviscosity, etc. as identified in reference (1).
It is known that the average state of polarization depends upon the cumulative nature of all of the plasma membranes of the cells in the population. The membrane of a cell is, fundamentally, a lipid bilayer in which protein molecules are embedded. This membrane play an important role in the metabolic activity of a cell in relation to its surroundings, since all interchanges between the cell and its surroundings must take place through the membrane. For this reason, the precise constitution of the membrane has been a most important object of cytological studies for a number of years. Reference (1) and a recent study reported in the Journal of Molecular Biology, volume 85, May, 1974, pp. 603-615, show that the state of polarization of fluorescence emitted by the cell population, (e.g., expressed in terms of the microviscosity of the entire population), is markedly different for normal and abnormal cell populations.
Useful conclusions can be drawn from the average state of polarization of fluorescence emitted by a population of cells. Were it possible to determine, accurately, the manner in which the state of polarization of fluorescence emitted by a cell is distributed throughout the cell population, as distinguished from the average state of polarization of the population as a whole, those cells in the population whose state of polarization differs from the population average could be quantified enabling conclusions to be drawn in evaluating the normality of the cell population. For example, the presence of malignant cells could be determined, or white blood cells could be differentially analyzed.
Microscope fluorometry is a technique for obtaining a measure of the state of polarization of fluorescence emitted by cells in a population; and is described in reference (2). In the conventional approach, cells labelled with a lipid soluble fluorescent dye are deposited on a slide operatively positioned relative to the objective of a microscope rigged to project a polarized beam of excitation radiation through the objective and onto a cell, or a portion of a cell, localized with respect to the objective. Associated with the objective is an imaging lens establishing a single output channel having a polarizer for polarizing the fluorescent output of the localized cell, and a rotatable analyzer for measuring the intensity of fluorescence polarized in directions parallel and perpendicular to the direction of polarization of the excitation beam. With this arrangement, the two output intensities are obtained sequentially. Consequently, the measurements will be sensitive to fluctuations in the intensity of the excitation beam, and the effect of time-dependence of the fluorescence itself due to bleaching of the dye, for example. In addition, inhomogeneities in the optical system with respect to polarization introduce further errors in the measured intensities. As a consequence, reliance cannot be placed on the measurements obtained, and definitive conclusions cannot be drawn.
It is therefore an object of the present invention to provide a new and improved method of and apparatus for determining the normality of cells wherein the deficiencies outlined above are overcome or substantially reduced.